Most normal somatic human cells cannot proliferate indefinitelyin vitro. After months of continuous culture and expansion,cells gradually and asynchronously cease division and entera permanent growth-arrested state called replicative senescence(1, 2)
. Senescent cells are viable and exhibit a number ofcharacteristic phenotypic and biochemical changes, includingan enlarged and flattened morphology, expression of SA-ß-gal3activity, and increased autofluorescence (1, 3, 4)
. Certainexogenous treatments can induce cells to rapidly acquire characteristicsof senescent cells. For example, early passage rodent or humanfibroblasts can be induced to undergo "premature" senescenceby expression of an activated ras oncogene (5)
. Other oncogenes,usually of viral origin, or somatic mutations allow human cellsto escape replicative senescence and continue to proliferateuntil they enter crisis in which proliferation is opposed byincreasing levels of cell death, and there is no net increasein cell number. These oncogene products are thought to bypasssenescence, at least in part, by neutralizing the tumor suppressorproteins p53 and p105Rb(6, 7)
. Clonal cell populations withan unlimited division potential may emerge from crisis in aprocess called immortalization.

Replicative senescence is, thus, the first barrier that preventsthe indefinite proliferation of cultured cells, and it appearsto be an important tumor suppressive mechanism in animals (8,9, 10)
. Cellular senescence may also underlie certain aspectsof organismal aging (11)
. Thus, there is considerable interestin determining the signal that causes cells to senesce and inunraveling the mechanisms responsible for executing the senescenceprogram. Several lines of evidence indicate that telomeraseand telomere length play an important role in replicative senescence.As normal somatic cells are passaged, telomeres generally shortenwith cell division, and senescent cells typically have shortertelomeres than do primary cells (12, 13, 14)
. In addition,primary and senescent cells in general express low levels oftelomerase, the enzyme responsible for synthesizing new telomericDNA at the ends of chromosomes. In contrast, most immortal andtumor cells maintain telomere length and express telomerase(12, 15, 16, 17, 18)
. These findings suggest that telomereerosion may be the mechanism by which cells record their replicativehistory, and that short telomeres may initiate replicative senescence(19, 20, 21, 22)
. Indeed, constitutive expression of an exogenoustelomerase gene that prevents telomere shortening allows theindefinite proliferation of some primary human cell types, suchas fibroblasts and retinal pigment epithelial cells (23, 24,25, 26, 27, 28)
. Immortalization of other cell types, suchas keratinocytes, requires both the expression of telomeraseand additional events, such as inactivation of the retinoblastomatumor suppressor pathway (29, 30)
. Conversely, inhibitionof telomerase activity in immortal or cancer cells leads totelomere shortening and eventual senescence or cell death (31,32, 33, 34)
.

The in vitro life span of human keratinocytes can be extendedby the high-risk HPV E6 and E7 oncogenes (35)
, although immortalizationappears to require additional as-yet-undefined events (30, 36). The high-risk HPV E6 protein binds to p53 and targetsit for accelerated ubiquitin-mediated degradation, and the high-riskHPV E7 protein binds to hypophosphorylated members of the retinoblastomafamily, resulting in their destabilization and the disruptionof Rb/E2F repressor complexes (37, 38, 39, 40, 41)
. In addition,the E6 protein induces telomerase activity in keratinocytes(42, 43)
. Genetic studies indicate that the ability of theE6 protein to activate telomerase is essential for keratinocyteimmortalization (29)
, and the reduction in telomere lengththat occurs in keratinocytes during passage prior to immortalizationis arrested or reversed in HPV-immortalized cells (36, 43)
.Furthermore, essentially all cervical carcinomas, which developfrom keratinocytes, and all cervical carcinoma-derived celllines constitutively express the E6 and E7 oncogenes from integratedHPV genomes (35)
.

We are exploring the consequences of repression of HPV E6/E7genes in cervical carcinoma cell lines, including HeLa cells,which contain HPV 18 DNA and harbor wild-type p53 and p105Rbgenes. This is accomplished by infecting cells with a SV40-basedrecombinant viral vector that expresses the BPV E2 regulatoryprotein, which binds the HPV early promoter and represses transcriptionof the E6 and E7 genes (44, 45, 46)
. This results in reactivationof the p53 and p105Rb tumor suppressor pathways and a rapidand profound growth arrest in the G1 phase of the cell cycle(44, 47, 48, 49, 50)
. The growth-arrested cells rapidly andsynchronously acquired numerous characteristics of primary cellsundergoing replicative senescence including changes in morphology,expression of SA-ß-gal, increased autofluorescence,and inhibition of telomerase activity (51)
. Similar resultswere obtained in the HT-3 (HPV30) and CaSki (HPV16) cervicalcarcinoma cell lines, as well as in freshly established cervicalcarcinoma cell lines containing HPV16 DNA, which suggests thatsenescence may be a general response to oncogene withdrawalin HPV-containing cells (51, 52)
. The E2 protein induced growtharrest only in cell lines containing HPV DNA, and constitutiveexpression of HPV E6/E7 prevented growth arrest and activationof tumor suppressor pathways (47, 49, 53)
. Transfection ofHeLa cells with an E2-expressing vector also generated cellsthat displayed senescent characteristics (54)
.

These data suggest that induced senescence in cervical carcinomacell lines is the result of a specific program that is activatedby repression of the HPV oncogenes. Repression of the E6 and/orE7 oncogenes may generate a senescent signal de novo. Alternatively,proliferating HeLa cells may contain a pro-senescent signal,but expression of HPV E6 and/or E7 prevents the cells from executingthe senescence program in response to this signal. Because ofthe importance of telomerase and telomere length in replicativesenescence, we decided to assess the role of these factors ininduced senescence in cervical carcinoma cells. The parentalHeLa cells used in these experiments express telomerase buthave short telomeres of about 2 kb pairs, a length shorter thanthat at which normal keratinocytes senesce but within the rangereported for different strains of HeLa cells (55)
. In addition,E2 expression caused a rapid reduction in telomerase activity(51)
. These observations are consistent with the hypothesisthat the loss of the HPV oncogenes allows the cells to respondto a telomere-based signal that initiates senescence. To testthis hypothesis, we expressed exogenous telomerase in HeLa cellsto generate cell clones with telomeres as long as those in proliferatingkeratinocytes and determined the response of these cells toexpression of the BPV E2 protein. The results of these experimentsindicate that increased telomere length and maintenance of high-leveltelomerase activity do not protect HeLa cells against growtharrest or senescence induced by HPV oncogene repression, whichimplies that induced senescence in cervical carcinoma cellsis not triggered by short telomeres or low-level telomeraseactivity.

Generation of HeLa Cells Expressing a Transduced hTERT Gene.
The experiments reported in this article were carried out totest whether the short telomeres in HeLa cells or the low levelof telomerase activity after E6/E7 repression was required forinduced senescence. For this purpose, we determined the consequencesof expressing the BPV E2 protein in HeLa cells that stably overexpressedthe catalytic subunit of human telomerase (hTERT) from a heterologouspromoter. We used infection with a recombinant SV40 viral vector,designated here the "E2 virus," to acutely express the BPV E2protein in a subcloned line of HeLa cells, HeLa/sen2 cells,which efficiently and synchronously undergo senescence in responseto E2 expression and E6/E7 repression (51)
. We chose to usethese cells because they display a very low background of proliferatingcells after E2 expression. Therefore, the growth-inhibited cellsare not overgrown by cells that escaped E2-induced senescence,and it is possible to analyze the phenotype of the arrestedcells for several weeks. However, it should be noted that E2-inducedsenescence occurs not only in HeLa/sen2 cells but also in unclonedHeLa cells and other cervical carcinoma cell lines containingHPV16 or HPV30 DNA.

The hTERT gene was transduced into HeLa/sen2 cells by usinga retrovirus containing the wild-type hTERT cDNA and a genecoding for resistance to G418 (LXSN-hTERT). Control cells weregenerated by infection with the empty LXSN retrovirus that containedthe drug resistance gene only. Individual G418-resistant colonieswere expanded into cell lines, and the lengths of their telomereswere determined. Genomic DNA was isolated and digested witha mixture of restriction endonucleases that do not cut withinthe (TTAGGG)n telomeric repeats. The digested DNA was resolvedby agarose gel electrophoresis, denatured, transferred to asolid support, and hybridized to a telomere-specific oligonucleotide.Fig. 1
displays the results obtained from representative LXSNand hTERT clones, as well as from the parental HeLa/sen2 cells.The broad smears are attributable to the heterogeneous telomerelengths present even in these recently cloned cell lines. Thetelomeres from the HeLa/sen2 cells and LXSN clones are extremelyshort, ranging from 1.5 to 2.5 kb in length, far shorter thanthe length at which primary keratinocytes are reported to senesce.None of the seven control clones established with the emptyvector exhibited an increase in telomere length relative tothe starting cells. In contrast, the transduced hTERT gene causeda dramatic though variable increase in average telomere lengthin 15 of the 20 clones examined (Fig. 1
and data not shown).The intense hybridization signals obtained with the hTERT cloneswith extended telomeres are attributable in large part to theincreased number of telomere repeats complementary to the probein these telomeres and to the relative compression of the largerDNA fragments in the upper portion of the gel. The two hTERTclones with the longest telomeres, hTERT-F and hTERT-J, as wellas the controls LXSN-1 and LXSN-4, were selected for furtheranalysis. These four clones had similar growth rates with anaverage doubling time of 26 h (data not shown). Telomerase activityin these hTERT clones was markedly increased compared with theparental cells (see Fig. 5
).

Fig. 1. Telomere lengths of cloned HeLa cell lines. Genomic DNA was prepared from parental HeLa/Sen2 cells (HeLa) and from clones generated with the LXSN vector or hTERT retroviruses. The DNA was digested with HinfI and RsaI, separated by electrophoresis, transferred to a nylon membrane, and probed with a telomere-specific oligonucleotide. The seven lanes on the left show the short telomeres present in the parental HeLa/Sen2 and the indicated LXSN cell lines. The eight lanes on the right show that the telomeres are considerably longer in the majority of the hTERT cell lines.

Fig. 5. Effect of the E2 protein on telomerase activity and telomere length. In A, protein extracts were prepared from mock (M) or E2-infected cells after 1, 2, or 8 days, as indicated, and telomerase activity was determined by a modified TRAP assay using 100 ng of LXSN-1 and 10 ng of hTERT-F cell protein. The lower band (C) represents the 36 nucleotide internal control that allows quantitative comparisons between different samples, and the upper bands (T) are the result of in vitro telomerase activity adding variable numbers of the six nucleotide telomere repeats. The shortest of these bands is a 50-nucleotide product containing three telomere repeats. The two lanes on the left are negative control reactions containing no extract (-) or 100 ng of heat-inactivated extract from LXSN-1 cells (HI). In B, a phosphorimager was used to quantitate the telomerase activity relative to the internal control, and the values were normalized to the amount of protein used in each assay. The results are shown on a log scale to facilitate the comparison between samples with markedly different telomerase activity. C, telomeres were visualized as in Fig. 1
for LXSN-1 and -4, and hTERT-F and -J either 2 days after mock infection or 8 days after infection with the E2 virus. For comparison, the telomeres in proliferating human foreskin keratinocytes (FK) and human cervical keratinocytes (CK) are also shown. In the first lane, the DNA size markers along with their size in kbp; numbers at the bottom of the other lanes, the modal telomere length in kbp for each sample.

Effect of hTERT on HPV Gene Expression and Tumor Suppressor Pathways.
We previously showed that infection of HeLa cells with the E2virus led to greatly reduced levels of the HPV18 E6/E7 mRNAs,followed by activation of the p53 and p105Rb tumor suppressorpathways. To determine whether the E2 protein had similar effectsin the hTERT-transduced cells, we first tested if expressionof the E2 protein repressed the endogenous HPV oncogenes. TheLXSN and hTERT clones were either mock infected or infectedwith the E2 virus, and RNA was prepared 1 and 2 days after infection.As shown in the Northern blot in Fig. 2A
, expression of theE2 protein caused rapid and complete repression of the HPV18E6/E7 genes in both the LXSN controls and hTERT clones. In bothtypes of cells, HPV expression remained repressed 8 days afterinfection (data not shown).

Fig. 2. HPV oncogene repression and activation of tumor suppressor pathways. In A, total RNA was isolated from the LXSN or hTERT cell lines 1 or 2 days after infection with the E2 virus, as indicated, or 2 days after mock infection (M). Five µg of RNA from each sample was electrophoresed in a formaldehyde denaturing gel, transferred to a nylon membrane, and hybridized to a 32P-labeled probe specific for the HPV18 E6/E7 genes. Only the major band is shown here, but all of the other bands exhibited identical regulation after E2 expression. In B, total protein extracts were prepared from LXSN-1 or hTERT-F cells 1, 2, or 8 days after infection with the E2 virus, as indicated, or 2 days after mock infection (M). Five µg of protein from each sample was subjected to denaturing gel electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane and probed with antibodies specific for p53, p21, p105Rb, or cyclin A. Hyperphosphorylated p105Rb corresponds to the upper band in the mock lane, and hypophosphorylated p105Rb is present in the lower band.

We next determined whether expression of hTERT affected activationof the p53 and p105Rb tumor suppressor pathways in responseto the E2 protein. The p53 pathway was monitored by accumulationof p53 and by the induction of one of its transcriptional targets,the cdk inhibitor p21sdi1/cip1/WAF1. Activation of the p105Rbpathway was monitored by the increase of hypophosphorylatedp105Rb and by repression of cyclin A, the transcription of whichis inhibited by the p105Rb-E2F complex. Protein extracts preparedfrom mock-infected cells or from cells 1, 2, and 8 days afterinfection with the E2 virus were analyzed by immunoblotting.Fig. 2B
presents the data from the LXSN-1 and hTERT-F celllines, which display the greatest difference in telomere lengths.In both control and hTERT cells, p53 levels were greatly increasedby 1 day after infection and then declined to mock-infectedlevels. p21 was also transiently induced in both cell linesalthough there was a slight delay in the hTERT-F cells. TheE2 protein also induces mdm2 expression (47, 51)
, which ispresumably responsible for the subsequent reduction in p53 and,in turn, p21 expression. In both cell lines, infection withthe E2 virus also resulted in a rapid but transient increasein the levels of the hypophosphorylated, active form of p105Rband a decrease in the hyperphosphorylated form, and cyclin Awas repressed. The other retinoblastoma family members, p107and p130, as well as several other E2F-responsive genes, respondedidentically to E2 expression in control and hTERT clones, despitethe presence of long telomeres in the latter cells (data notshown). The LXSN-4 and hTERT-J lines also responded to E2 expressionby transiently activating the p53 and retinoblastoma tumor suppressorpathways to a similar extent and with similar kinetics (datanot shown). Thus, increased telomerase activity and extendedtelomeres did not affect the induction of tumor suppressor pathwaysafter repression of HPV oncogene expression.

HeLa Cells Expressing Telomerase Remain Sensitive to E2-mediated Growth Arrest and Senescence.
Because expression of the E2 protein repressed HPV18 E6/E7 expressionand activated the p53 and p105Rb pathways in the hTERT clones,we determined its effect on the cellular phenotype. The transducedclones were infected with the E2 virus, and cellular DNA synthesiswas determined 2 days later by measuring the incorporation oftritiated thymidine (Table 1)
. As expected, after infectionwith the E2 virus, the control LXSN clones incorporated onlyabout 3% as much thymidine as did mock-infected cells. Expressionof the E2 protein reduced DNA synthesis to a similar extentin hTERT clones, indicating that increased telomerase activityand longer telomeres offered no protection from the inhibitionof DNA synthesis.

Although hTERT expression did not prevent E2-induced growtharrest, it remained possible that the arrested cells were notsenescent. Therefore, we examined the phenotype of the arrestedhTERT cells to determine whether they underwent senescence.Eight days after mock infection or infection with the E2 virus,cellular morphology was determined by phase-contrast microscopy(Fig. 3A)
. The mock-infected cells formed discrete, compactcolonies of small cells. The E2 virus-infected cells did notform colonies. Instead, they failed to form tight cell-cellcontacts, became motile, adopted an elongated morphology, andappeared larger and more granular, resembling the morphologyof senescent cells. Importantly, the LXSN and hTERT cells respondedidentically to E2 expression.

Fig. 3. Cellular morphology and SA-ß-gal activity. A, phase micrographs at x200 show colonies of LXSN-1 and TERT-F cells 8 days after mock infection (top panels) or the isolated cells 8 days after infection with E2 virus (bottom panels). Increased size, flattening, granular cytoplasm, and lack of intercellular contacts are observed in the treated cells. In B, LXSN-1 or hTERT-F cells were stained for SA-ß-gal activity 15 days after mock-infection (top panels) or infection with E2 virus (bottom panels) and were photographed using brightfield optics at x200.

Cells that have undergone replicative senescence show increasedintrinsic fluorescence, called autofluorescence, which is thoughtto be caused by the accumulation of oxidatively damaged proteinsand lipids. LXSN-1 and -4 cells and hTERT-F and -J cells wereeither mock infected or infected with the E2 virus. After 8days, the cells were harvested, and autofluorescence was determinedby flow cytometry. All four of the cell lines responded identicallyto infection with the E2 virus (Fig.4
and data not shown).Specifically, E2 expression induced the entire population ofcells to uniformly shift to higher levels of autofluorescence,ruling out the possibility that a subpopulation of the transducedcells escaped senescence but was masked by greater numbers ofsensitive cells. Taken together, these results indicate thatsenescence is still induced by HPV repression in the hTERT-transducedHeLa cells that harbored greatly increased telomere lengths.

Fig. 4. Effect of the E2 protein on autofluorescence. LXSN or hTERT cell lines were either mock infected or infected with the E2 virus, and cells were collected by trypsinization 8 days later. The cell suspension was analyzed by flow cytometry as described in the "Materials and Methods" section. Histograms of the log of the autofluorescent signal are shown for mock-infected or E2-infected samples, as indicated.

Effects of the E2 Protein on Telomere Length and Telomerase Activity.
To determine the effects of the transduced hTERT genes on telomeraseactivity, we assayed telomerase activity in extracts preparedat various times after infection with the E2 virus or mock infection.As shown in Fig. 5A
, 100 ng or 10 ng of protein from the LXSNor hTERT cell lines, respectively, were subjected to an in vitroTRAP assay, and the amplified products were separated on a nondenaturingpolyacrylamide gel. Because of the high amount of telomeraseactivity in the hTERT cell lines, 10 ng of protein from thesecells were assayed to maintain the linearity of the assay. "C"marks the position of the internal control amplification productthat allows quantitative comparison between the samples, whereasthe intensity of the more slowly migrating ladder of bands isa measure of telomerase activity. HeLa cells and LXSN controlscontained readily detectable telomerase activity, and extractsof uninfected hTERT-F and -J lines exhibited about 50-fold moretelomerase activity than did the LXSN-1 samples (Fig. 5A)
.The sample containing heat-inactivated protein extract and thesample without extract did not display activity, demonstratingthe specificity of the assay. As was the case for the parentalHeLa/sen2 cells, telomerase activity rapidly declined in boththe LXSN clones and in the hTERT-transduced cells after infectionwith the E2 virus. Fig. 5B
shows the results normalized tothe amount of protein used in the TRAP assay to allow readycomparison between the LXSN-1 and hTERT-F samples. This analysisrevealed that despite the reduction in activity after infection,the absolute levels of telomerase activity of the senescenthTERT-F cells 8 days after infection were still greater thanthe activity of proliferating uninfected LXSN-1 cells. A Northernblot demonstrated that the levels of mRNA from the transducedhTERT gene did not decline over the 8 days after infection withthe E2 virus, which indicated that the decline of telomeraseactivity was posttranscriptional (data not shown).

We also determined telomere lengths of mock-infected and E2-infectedcells 8 days after infection and compared them with those ofproliferating primary keratinocytes (Fig. 5C)
. Infection withthe E2 virus and the induction of senescence had no effect onthe length of the telomeres. For comparison, the telomere lengthsof primary keratinocytes from two different sources are alsoshown. DNA was purified from early passage foreskin and ectocervicalkeratinocytes that were not yet senescent, and the telomerelengths were determined. Both types of keratinocytes had telomeresthat were much longer than those of the parental HeLa/sen2 cellsor LXSN clones. The hTERT clones have telomeres that are longerthan those in proliferating ectocervical keratinocytes, andthe hTERT-F telomeres are about the same length as those presentin newborn foreskin keratinocytes. Thus, although the hTERTclones possess telomeres as long as, or longer than, those inactively proliferating keratinocytes, they readily underwentE2-mediated senescence, as did control HeLa/sen2 cells, whichcontain very short telomeres.

There is considerable interest in studying the molecular basisof cellular senescence because of its potential role in organismalaging and tumor suppression. The correlation between replicativesenescence and telomere erosion led to the formulation of thetelomere hypothesis, which proposes that the gradual reductionin telomere length cause by successive rounds of DNA replicationand cell division causes the length of one or more telomereslength to fall below a certain threshold, resulting in the initiationof senescence (19, 20, 21, 22)
. The strongest evidence in supportof this hypothesis is the finding that ectopic expression oftelomerase and extension of telomeres in primary human cellscan delay or prevent senescence (24, 25, 28)
. Furthermore,in the case of human keratinocytes, the ability of the high-riskHPV E6 protein to induce telomerase activity appears importantfor extending the replicative life span of the cells and inducingimmortalization in combination with HPV E7 or other events thatinactivate the retinoblastoma pathway (29)
. However, thereare situations in which short telomeres are not sufficient totrigger cell senescence. For example, in human fibroblasts expressinghTERT or human keratinocytes expressing HPV 16 E6/E7, telomeresstabilize at lengths even shorter than those in cells undergoingcrisis (36, 43, 56)
. Furthermore, many cancer cells, includingthe uninfected HeLa cells studied here, harbor short telomeresbut, nevertheless, actively proliferate (55, 57, 58)
.

Although replicative senescence was originally defined as occurringafter extended passage of primary cells, several exogenous treatmentscan cause early-passage cells to rapidly acquire various phenotypescharacteristic of senescent cells. Such premature senescencecan be triggered in primary fibroblasts by the introductionof activated ras alleles and activation of the raf/mitogen-activatedprotein (MAP) kinase signaling cascade; sublethal levels ofhydrogen peroxide or DNA-damaging agents; high-level expressionof cdk inhibitors, E2F1, p14ARF, p105Rb, or p53; or treatmentwith other chemical or physical agents (e.g., as seen in Refs.5
and 59, 60, 61
). In these situations, the cells undergosenescence long before they would have reached the senescencebarrier during continuous passage. Because these early-passagecells contain relatively long telomeres, and senescence ensuedtoo rapidly for telomeres to undergo significant shortening,it appears that premature senescence is independent of telomerelength. Indeed, introduction of the hTERT gene did not blockras-induced senescence (62)
. These results imply that diversestimuli can initiate the senescent program in early-passagecells.

The senescent phenotype can also be elicited in immortal orcancer cells by expression of the tumor suppressors p53, p105Rb,p27KIP, p21, or p16INK4(63, 64, 65, 66, 67, 68, 69, 70, 71)
,by thermal inactivation of a temperature-sensitive SV40 largeT antigen (72, 73, 74)
, by treatment with transforming growthfactor ß (75)
or chemotherapeutic agents (76)
, or,as reported here and elsewhere, by repression of the HPV oncogenesin cervical carcinoma cell lines (51, 52, 54)
. In contrastto early-passage cells undergoing premature senescence, thesecells have proliferated far beyond the senescence barrier, and,in many cases, telomere erosion has already occurred, and thecells harbor short telomeres. Indeed, telomeres in the parental,proliferating HeLa cells are far shorter than they are in early-passagehuman keratinocytes, and HPV E6/E7 repression causes a rapiddecline in telomerase activity. These results suggested thatexpression of the HPV E6 and/or E7 proteins prevented transductionof the senescent signal from the short telomeres in the cells,and that once E6/E7 expression was extinguished, the cells sensedthe presence of short telomeres and initiated the senescenceprogram. Alternatively, the low level of telomerase after E6/E7repression may signal senescence. To test these possibilities,we generated HeLa cell clones with increased telomere lengthand elevated telomerase activity. Elimination of HPV E6 andE7 expression in these HeLa cell derivatives resulted in theacute activation of the p53 and retinoblastoma tumor suppressorpathways and the rapid acquisition of the senescent state. Thus,the presence of long telomeres and active telomerase did notprotect HeLa cells from induced senescence after E6/E7 repression.

It is unclear whether the level of telomerase or the lengthof telomeres controls replicative senescence. In normal humanoral keratinocytes, onset of replicative senescence correlatesbetter with telomerase levels than with telomere length, andin other systems, telomerase expression, rather than telomeremaintenance, appears important for the bypass of senescence(23, 77)
. However, in other cell types, telomere maintenanceis a better predictor of proliferation than is telomerase activity(27, 78)
. The results reported here indicate that the proximalsignal that induces senescence in HeLa cells is neither shortaverage telomere length nor a low absolute level of telomeraseactivity. However, these results do not rule out a role fortelomerase or telomere length in induced senescence. It is possiblethat a particular telomere remains short in the hTERT-expressingcells, although the average length of telomeres is greatly extended,or that the cells sense the decline in telomerase activity,rather than absolute activity. Alternatively, short telomeresmay affect the transcription of telomeric genes involved insenescence or induce structural chromosomal abnormalities. Ifthese changes persist despite the reexpression of telomerase,they may initiate senescence when the HPV oncogenes are repressed.

It is also possible that E6/E7 repression can initiate a senescenceprogram independently of telomerase and telomere length. E6/E7repression induces expression of p53, p21, and hypophosphorylatedp105Rb, all of which have been implicated in inducing senescencein various cell systems. In the E2 transfection experimentsof Wells et al., p21 was proposed to mediate induced senescence(54)
. However, E2-expression also induces senescence in p53-minusHT-3 cervical carcinoma cells in the absence of p21 induction(49, 51)
, which indicates that the induction of p53 and p21is not required for senescence in this system. p16INK4 and activatedoncogenes can also induce senescence in some settings. Expressionof p16INK4 is constitutively high in HeLa cells and persistsduring induced senescence (51)
; therefore, repression of E6/E7may allow the cells to undergo senescence in response to theelevated levels of p16INK4. HeLa cells may also contain activatedoncogenes, which may induce senescence once E6/E7 are repressed.Experiments are currently underway to test these possibilities.

The rapid and synchronous onset of induced senescence is inapparent contrast to the long time required for replicativesenescence to occur during continuous passage of primary cells,raising the possibility that induced senescence is mechanisticallydistinct from replicative senescence. Moreover, expression ofexogenous telomerase and telomere extension do not prevent inducedsenescence, in contrast to the protection that telomerase expressionaffords against replicative senescence. However, cells undergoingreplicative and induced senescence display indistinguishablephenotypes, which suggests that these processes share a mechanisticbasis. In fact, it is possible that an individual cell undergoingreplicative senescence during passage progresses rapidly throughthe senescent program. According to this view, the slow andasynchronous nature of replicative senescence results from theheterogeneous length of time before different cells enter thispathway. This heterogeneity may arise from the different lengthsof telomeres in the starting cell population, or from cell-to-cellvariability in generation time, or the rate of telomere shorteningduring passage. In the case of induced senescence, this programis initiated synchronously in all of the cells.

HPV E6 expression induces telomerase activity in early passagekeratinocytes, an effect that is thought to be attributableto E6-mediated activation of the hTERT promoter (42, 43, 79, 80). Telomerase activity declined in hTERT cells infected withthe E2 virus, although the hTERT gene was expressed from a retroviralpromoter, and there was no apparent loss of hTERT mRNA expressedby the transgene. This result implies that E2 expression andHPV E6/7 repression exert a posttranscriptional effect on theexpression or activity of telomerase. The basis of this effectis currently unknown, but it may reflect the down-regulationof telomerase activity induced by growth arrest or by p105Rbexpression in other systems (16, 71)
.

We previously suggested that induction of senescence in cervicalcarcinoma cells by inhibition of the expression or activityof the HPV oncogenes may represent a novel therapeutic approachfor this cancer (51)
. The results reported here suggest thatthis approach may not be restricted to those cancers with shorttelomeres. The pathway leading to induced senescence in cancercells warrants further study, because manipulations that activatethis pathway may limit the in vivo growth of many types of cancers,including those of nonviral etiology.

Cell Lines and Viruses.
HeLa/sen2 cells are a subclone of HeLa cells that respond rapidlyand uniformly to infection with the E2 virus (51)
. The LXSNcontrol retrovirus was obtained by transfecting amphotropicBing packaging cells with the pLXSN vector and collecting acell-free supernatant 2 days later. A cell line producing LXSN-hTERTretrovirus was kindly provided by Denise Galloway (Fred HutchinsonCancer Research Center, Seattle, WA). The hTERT cDNA was clonedfrom a HeLa cell library, inserted into pLXSN, and packagedusing the PG13 cell line.4
Retrovirus-containing supernatantswere used to infect HeLa/sen2 cells at a low multiplicity, andtransduced cell lines were cloned from individual colonies after2 weeks of selection in 1 mg/ml G418. Cells were maintainedin complete media with 0.5 mg/ml G418, but G418 was omittedduring all of the experiments.

High titer stocks of the recombinant SV40 virus expressing theBPV E2 protein (pPava-5'BS-RMC, designated the E2 virus) wereprepared, titered, and used to infect cells at a multiplicityof 20 as described previously (47, 49)
. For later time points,a second infection at the same multiplicity was done at 3 daysto reduce the background attributable to the outgrowth of cellsthat escaped the first infection. The growth medium was changedevery 3 days. For biochemical analysis, the HeLa-derived celllines were seeded in 150-mm dishes and infected the followingday. Washed cells were scraped and collected into aliquots bycentrifugation and stored as frozen cell pellets until processing.

Telomere Length.
Genomic DNA was prepared from cell pellets using a Qiagen DNeasykit including the RNase digestion according to the manufacturersinstructions. One µg of genomic DNA was then digestedwith RsaI and HinfI and resolved by electrophoresis in a 0.7%agarose gel with DNA size markers. The DNA was partially depurinatedin 0.25 M HCl for 30 min and was then fragmented and denaturedin 3 M NaCl- 0.4 M NaOH for 30 min. The gel was equilibratedin 3 M NaCl-8 mM NaOH; the DNA was transferred to a Nytran Superchargemembrane (Schleicher and Schuell) and neutralized in 0.2 M sodiumphosphate buffer (pH 6.8). The DNA was UV-cross-linked to themembrane with a Stratalinker 1800 (Stratagene), prehybridizedin 5x SSPE [1x SSPE is 150 mM NaCl, 10 mM sodium phosphate (pH7.4), and 1 mM EDTA], 5x Denhardts solution (Sigma ChemicalCo.; 50x Denhardts is a 1% solution of BSA, ficoll, andpolyvinyl pyrrolidone), and 0.5% SDS for 1 h at 37°C. Then15 pmol of the 32P-5' end-labeled, (TTAGGG)3 telomere-specificoligonucleotide was added, and the mixture was hybridized overnight.The membrane was washed three times in 2x SSPE-0.5% SDS at roomtemperature and the bands visualized with a phosphorimager.The membrane was subsequently hybridized with random-primed-labeledprobes for the DNA size markers to estimate the lengths of thetelomeres.

Cell Analysis.
DNA synthesis was measured in quadruplicate by incorporationof [3H]thymidine. Cells (2.5 x 104) were seeded per well ina 24-well dish and were infected with the E2 virus the nextday. Two days later, the labeling and measurement of incorporationwere performed as described previously (51)
. For determinationof cell morphology and SA-ß-gal activity, 5 x 104or 4 x 102 cells were seeded in 35-mm wells for experimentaland mock-infected samples, respectively. The cells were infectedor mock infected the next day and again 3 days later, and subsequentlywere refed every 3 days. Cells were photographed through a phasemicroscope 8 days after the first infection, to document changesin morphology. SA-ß-gal activity was determined atpH 6.0 (3)
15 days after the initial infection with the E2virus- or mock-infection and documented by photography withbrightfield optics.

For flow cytometry, 2 x 105 cells were seeded per 100-mm dish,infected the following day with the E2 virus. Matched cellsfor mock infection were seeded at 2 x 104 per dish and maintainedin parallel. The cells were harvested by trypsinization after8 days, washed with complete medium, washed once at 4°Cwith PBS and resuspended in PBS at 1 x 106 cells/ml. Data werecollected using a FACSCalibur flow cytometer (Becton Dickinson,San Jose, CA) with CellQuest software. After excitation at 488nm, autofluorescence was measured after passage through a 530/30-nmband pass filter and listmode data files were analyzed withWinMDI 2.8 software. At least 20,000 cells were analyzed foreach sample.

Biochemical Analysis.
LXSN or hTERT cell lines were seeded in 150-mm dishes and infectedthe following day. Washed cell pellets were frozen at varioustimes after infection, and immunoblot analyses of total extractedprotein were carried out as described previously (51)
withthe following primary antibodies: 15801A (p53) and 14001A (p105Rb)from PharMingen; sc-397 (p21), sc-318 (p107), sc-317 (p130),and sc-8432 (pan-actin), from Santa Cruz Biotechnology; andanti-cyclin A, a gift from H. Zhang (Yale University, New Haven,CT). After washing, filters were incubated with a 1:20,000 dilutionof species-specific donkey antibody conjugated with horseradishperoxidase (Jackson ImmunoResearch). Immunoblots were incubatedwith ECL+Plus (Amersham), and the signals were detected usingHyperfilm (Amersham).

Total RNA was prepared by using the RNeasy Mini kit (Qiagen).RNA was denatured, resolved on a 1% agarose-formaldehyde gel,transferred to Nytran Supercharge (Schleicher and Schuell),and cross-linked to the membrane using a Stratalinker 1800 (Stratagene).mRNAs were detected by hybridization with random-prime labeledprobes.

Telomerase activity was assayed in protein extracts preparedat various times after infection with the E2 virus by usinga TRAPeze Telomerase Detection kit (Intergen, Purchase, NY).The reaction products were resolved on 6% polyacrylamide minigeland were detected and quantitated using a Storm 840 (MolecularDynamics, Inc.). The signal from the entire ladder of bandsattributable to telomerase activity was normalized to the signalfrom the internal control band and to the amount of cellularprotein analyzed. We previously showed that the signal was proportionalto the amount of extract assayed (51)
.

Acknowledgments

We thank Denise Galloway for providing the retrovirus expressinghTERT.

Footnotes

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